Flaviviruses are a group of positive-stranded RNA viruses that have a global impact due to their widespread distribution and ability to cause disease in humans and economically important domesticated animals. Several members of this genus, such as dengue virus (DENV) and West Nile virus (WNV), are considered emerging or re-emerging pathogens because the incidence with which they encounter humans and cause disease is increasing each year at an alarming rate.[unreadable] [unreadable] WNV is a mosquito-borne member of this genus and is the etiologic agent of West Nile encephalitis. WNV is endemic in parts of Africa, Australia, Europe, Asia, and the Middle East and has been responsible for periodic outbreaks of encephalitis in humans and horses. The introduction of WNV into North America in 1999 and its rapid spread across the United States into Canada, Mexico, and the Caribbean identifies this virus as an emerging pathogen of clinical and economic significance for the Western Hemisphere. While seroprevalence studies indicate that most WNV infections of humans are subclinical, clinically apparent infections range from a febrile illness (West Nile fever) to more severe and potentially fatal neurologic disease. Currently, no WNV vaccine has been approved for use in humans and treatment is supportive. [unreadable] [unreadable] Cryo-electron microscopy studies reveal that the surface of flavivirus virions is covered by a highly ordered icosahedron composed of 180 envelope proteins. The generation of antibodies capable of binding to this array of viral proteins and blocking infection is a critical aspect of the immune response and an important goal of vaccine development. Passive immunization studies and experimental infections of immunodeficient mice demonstrate that antibody plays a significant role in protection from flavivirus infection. The importance of antibodies in vivo reflects their ability to directly neutralize virus infectivity, facilitate complement-mediated lysis of virions, and promote efficient viral clearance through Fc-receptor dependent pathways. However, antibodies generated in response to natural infection or vaccination also have the potential to enhance virus infection both in vitro and in vivo. [unreadable] [unreadable] The neutralization potential of an antibody is governed by the number of sites on the virion available for binding (epitope accessibility) and the strength of binding (avidity). Using neutralizing antibodies that bind structurally distinct sites on the WNV virion, we are investigating the biochemical basis of potency with respect to how antibodies engage the virus particles, and in what numbers. Although molecular modeling studies of the mature virion suggest that many of the known epitopes on the E protein should be poorly accessible or completely cryptic, antibodies to these determinants still neutralize infection to varying degrees. To investigate mechanisms that govern the potency of antibodies that target cryptic epitopes, we are investigating the dynamics that control epitope accessibility and neutralization potency.[unreadable] [unreadable] Paradoxically, antibodies may also play a role in enhancing virus infection and exacerbating disease. Antibody-dependent enhancement of infection (ADE) describes a dramatic increase in infection of Fc-receptor-bearing cells in the presence of sub-neutralizing concentrations of antibody or immune sera. The most direct link between ADE and the clinical outcome of DENV infection comes from investigations of the unusually large number of DHF cases following primary infection observed in infants during the first year of life. At birth, DENV-specific passively acquired antibodies are present at a relatively high concentration and exhibit neutralizing activity in vitro. However, as the child ages, degradation of maternally acquired antibody continues to levels that are no longer protective, do not neutralize virus, and enhance virus infection in vitro. The waning antibody titers of infants to levels that support ADE in vitro parallels the risk of DHF following primary DENV infection during the first year of life. In a broader context, antibodies elicited by primary infection with one serotype of DENV may bind related viruses introduced during secondary infection with reduced avidity, resulting in engagement of the virion with a stoichiometry that does not permit virus neutralization but can support ADE. The development of an immune response that elicits protective levels of neutralizing antibodies against all four serotypes of virus present in the vaccine is a key factor for reducing the risk of ADE. To facilitate this goal, our laboratory is investigating the biochemical determinants that comprises the enhancing character of an antibody, and studying the cell biology that underlies the mechanism of ADE.